Dr. Zhu has the long-term goal of pursuing independent investigation in the field of allergic diseases. Receipt of a Mentored Clinical Scientist Development Award will facilitate the growth of Dr. Zhu's investigative skills by expanding his knowledge of molecular techniques, as outlined in the proposed studies. The learning objectives set out in this proposal, combined with the support of his Mentors, Drs. Alan R. Left and Kimm J. Hamann, and the critical environment within the Section of Pulmonary and Critical Care Medicine, and Ben May Institute for Cancer Research at the University of Chicago, will foster Dr. Zhu's progression to independent lines of investigation into the mechanisms of asthma. The central hypothesis of this proposal is that adhesion of eosinophils mediated by Beta2-integrin is regulated by cytosolic phospholipase A 2 (cPLA2) activation that is initiated by 1) Ras and 2) phosphoinositide 3 kinase (PI3K) induced extracellular signal-regulated kinase (ERK) activation. Three immediate goals are defined: (1) Determine the role of Ras in eosinophil adhesion. To test the hypothesis that Ras activation is required for eosinophil adhesion, we will determine the effects of Ras inhibition on IL-5 or eotaxin- stimulated eosinophil adhesion and CD1 lb avidity change, using pharmacological inhibitors and a dominant negative TAT-Ras fusion protein. Transfection of constitutively active Ras into eosinophilic AML14.3D10 cells also will be used to test whether Ras activation is sufficient for adhesion. (2) Determine the role of MAPK isoform activation in eosinophil adhesion. To test the hypothesis that Ras-mediated activation of ERK is essential for eosinophil adhesion, we will determine the effects of MAPK isoform inhibition on eosinophil adhesion using selective MAPK pathway inhibitors and dominant negative and active mutants of MEK1. Using dominant negative Ras and ERK pathway inhibitors, the causal relationship between Ras, ERK and cPLA 2 activation will be elucidated. (3) Determine the role of PI3K activation in eosinophil adhesion. To test the hypothesis that PI3K is required for eosinophil adhesion, we will determine the effects of PI3K inhibition on IL-5 or eotaxin- stimulated eosinophil adhesion and ERK phosphorylation using pharmacological inhibitors and a dominant negative TAT-PI3K fusion protein. Transfection of active PI3K into AML14.3D10 cells will be used to test whether PI3K activation is sufficient for adhesion. Knowledge of the signaling components required for eosinophil adhesion may provide insight into the pathogenesis of airway inflammation and lead to therapeutic interventions for human asthma.